The proposed project will examine the structure of two of the key regulatory enzymes of carbohydrate metabolism, phosphofructokinase (PFK) and fructose-1,6-bisphosphatase (Fru-P2ase), and integrate that information into the framework of knowledge concerning regulatory mechanisms. This research, which involves the collaborative effort of three laboratories, will include the following: (1) The complete covalent structure of rabbit skeletal muscle PFK and pig kidney Fru-P2ase will be determined. (2) Functionally important sites within PFK, including thiol groups that monitor conformational events, the lysine at the citrate binding site, and residues at both the AMP site and the MgATP inhibitory site, will be identified. (3) The structure of the sites of PFK and Fru-P2ase that are phosphorylated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase will be determined. The structures will be compared with that around the naturally occurring serine-P in PFK. (4) Patterns of secondary and tertiary structure will be sought on the basis of predictive analysis of the sequences. The structures will be integrated into the developing knowledge of the X-ray structure of these two enzymes. These studies will provide structural correlates of our considerable knowledge of the role of effectors of the activity of these enzymes and will be of interest not only in the specific case of these two pacemakers but also in the overall picture of allosterism.